Isolation And Identification Of Proteins From The Peritrophic Membrane In Loxostege Sticticalis

The meadow moth,Loxostege sticticalis L.,is an important agricultural pest in the north (North China; the Northeast; the Northwest) of China. It has broken out three times since 1949,which lead to enormous loss on agricultural and animal husbandry. Because of the properties of periodicity,the fundamental research on Loxostege sticticalis L. is unsubstantial. As the first crucial barrier for insects, peritrophic membrane(PM)has gradually been the hotpoint of the latest research of pest control. Based on the constructed cDNA library of the meadow moth midgut, we used PM proteins polyclonal antibody to isolate and characterize the related coding genes. Main achievements summarized as follows:RNeasy Mini Kit,Oligotex mRNA kit,ZAP-cDNA? synthesis kit and ZAP-cDNA? Gigapack? III Gold Cloning Kit were used to extract total RNA from larval midgut, then purify mRNA and construct cDNA library. Original library titer was 3.73×106pfu/ml, the recombinant rates of the blue-white spot screening were up to 99.7%, the average inserted gene fragment length was about 1.7kb.The 282 positive colonies were obtained by PM proteins polyclonal antibody of Spodoptera exigua immunoscreening from meadow moth midgut cDNA library. Six proteins were isolated and identified. The appropriate primers were employed to clone the full length of LstiCBP gene using RACE technique because of 5'-terminus incompleteness. GenBank accession No. of LstiCBP gene was FJ408730, contained an open reading frame(ORF) 2,403bp and encoded 801 amino acids, 15 of which composed the signal peptide. The precursor of LstiCBP protein was 86.2kDa and contained 8 chitin binding domains (CBDs). The successful prokaryotic expression was performed when LstiCBP recombined into pET30 vector. The Western blotting analysis demonstrated that LstiCBP existed in PM, midgut tissue, hemolymph, integument and ecdysis, especially rich in midgut. The amount of LstiCBP in PM was affected by M2R. Lsti99 and Lsti201 were still unknow proteins until now while the proteins were detected in head, PM, midgut tissue, hemolymph and integument of larvae. GenBank accession No. of gene Lsti99 was FJ798745, contained an ORF of 1,245bp and encodied 415 amino acids. The molecular weight of the precursor, which contained a 17-amino-acid signal peptide, was 47.1kDa. The Lsti99 successfully expressed in Escherichia coli after being recombined with pET30 vector, but couldn't be purified by Ni-NTA. The gene of Lsti99 was inserted to V5-His6 tagged baculovirus by GatewayTM, then Lsti99 protein was successfully expressed after recombined baculovirus transfecting the cell Sf9. The accession number in GenBank for gene Lsti201 was FJ798746. The cDNA was 2,145bp, and the longest open reading frame coded for 715 amino acids. The predicted precursor protein contained a signal peptide of 17 residues and 59 functional domains with a particular domain of Thr-rich area. The expressed protein was shown as 80.5kDa in E.coli.by SDS-PAGE analysis. The gene LstiSLC25, whose ORF was 897bp, encoded 299 amino acids. LstiSLC25 protein was a typical solute carrier with molecular weight 32.1kDa and realized the protein expression in prokaryotic cell. Carboxypeptidase was an important digestive enzyme in insect midgut, which had three compositions: signal peptide, propeptide and the enzyme. LstiCPA was 1 380 bp in full-length (GenBank accession no. EU924506), and the ORF encoded 434 amino acids, with the predicted MW 49.1kD and pI 9.56 respectively. The activity for Carboxypeptidase was 0.0005 when it expressed in E.coli.. Carboxylesterase was an important detoxificative enzyme of insect midgut. The GenBank accession No. of gene LstiCarE was EU339106. The open reading frame of LstiCarE was 1875bp and encoded 625 amino acids with the precursor's molecular weight 69.0kDa. It possessed the typical characters of carboxylesterase and had been successfully expressed in E.coli.There were at least 19 proteins in PM and most of them were lower than 94kDa. Feeding the larva with M2R could affect the types and the contents of the PM protein. The normal PM surface was smooth without pores and slits while the treated PM had pores and slits. The higher concentration and longer treatment, the more damage could cause to PM. Once M2R were added to Bt, the treated larva not only showed obviously shorter time of Bt toxicity to Loxostege sticticalis, but also reduced Bt using concentration.By isolating and identifying the PM proteins of meadow moth, we could get a clear idea of the physiological function toward the target site of PM. Through investigating the interaction mechanism between PM and pathogenic microorganism, we could hunt for novel target site and explore high efficient biological pesticide.